Biomod/2014/HKBUteam/experimentalresults

From OpenWetWare

Jump to: navigation, search


Contents

1st Trial

1. Conjugation condition

Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1. Mixtures were vortexed at room temperature for 4 hrs.

In-Complete route Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.


2. Mass-spectrometry

i. Control Group:

Fig.1(A)Control group one: MS-Single stranded DNA A2(B)Control group two: MS-Single stranded DNA A3
Fig.1(A)Control group one: MS-Single stranded DNA A2
(B)Control group two: MS-Single stranded DNA A3

ii. Conjugate Group:

Fig.2(A)For MS-single stranded DNA A2 ,a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.(C)For MS-single stranded DNA A6 , a peak at 11171 shows the conjugation of A6 and carbon nanodots
Fig.2(A)For MS-single stranded DNA A2 ,a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.
(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.
(C)For MS-single stranded DNA A6 , a peak at 11171 shows the conjugation of A6 and carbon nanodots


3. DNA extraction after folding of DNA origami

Gel photo :

(Fig.3, GeneRuler DNA Ladders, Samples, Modified Primers A2, A3)


4. Nanodrop Reading

DNA concentration :
0.8 ng/ul
260/280 : 1.34
260/230 : 0.86


5. Confocal Microscopy

Fig.4(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control (Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm) The intensity for the fluorescence is very low. It is interpreted that a minimal amount of drug has entered the cells which may due to low concentration of drug in condition of 1st trial.
Fig.4(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control
(Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm)
The intensity for the fluorescence is very low. It is interpreted that a minimal amount of drug has entered the cells which may due to low concentration of drug in condition of 1st trial.



2nd Trial

1. Conjugation condition

Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1. Mixtures were vortexed at room temperature for 4 hrs
In-Complete route Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.


2. Mass-spectrometry

i. Control Group 1 & 2 refer to Trial 1 Fig.1
ii. Conjugated Group:

Fig.5(A)For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.
Fig.5(A)For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.
(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.



3. DNA extraction after folding of DNA origami

Gel photo :

(Fig.6, GeneRuler DNA Ladders, Samples, Modified Primers A2, A3)


4. Nanodrop Reading

DNA concentration :
2.3 ng/ul
260/280 : 1.28
260/230 : 0.52


5. Confocal Microscopy

Fig.7(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control (Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm) The intensity for the fluorescence is enhanced with respect to 1st Trial. More drugs were entering the cell this time.
Fig.7(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control
(Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm)
The intensity for the fluorescence is enhanced with respect to 1st Trial. More drugs were entering the cell this time.

3rd Trial

1. Conjugation condition

Single strand DNA, A2, A3 and A6 conjugated with chlorambrucil, folic acid and carbon nanodots respectively in ratio 1:10. Mixtures were vortexed at 37 for 4 hrs. Complete route
All substituent was conjugated and folded simultaneously


2. Mass-spectrometry

i. Control group 1 & 2 refer to Trial 1 Fig.1
ii. Conjugated group

Fig.8(A)For MS-single stranded DNA A2 , a peak at 10193 shows the conjugation of A2 and chlorambrucil.(B)For MS-single stranded DNA A3 , a peak at 7043 shows the conjugation of A3 and folic acid.
Fig.8(A)For MS-single stranded DNA A2 , a peak at 10193 shows the conjugation of A2 and chlorambrucil.
(B)For MS-single stranded DNA A3 , a peak at 7043 shows the conjugation of A3 and folic acid.


3. DNA extraction after folding of DNA origami

Gel photo :

(Fig.9, GeneRuler DNA Ladders, Samples)


4. Nanodrop Reading

DNA concentration :
5.5 ng/ul
260/280 : 2.65
260/230 : 1.12


5. Confocal Microscopy

Fig.10(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control (Excitation : 405nm, Emission : 460 – 560nm) The intensity for the fluorescence is further enhanced with respect to 1st and 2nd Trial. The signal peaks at 473nm. A lot of drugs were entering the cell. It may due to the enhancement done to the reaction condition of drugs.

Fig.10(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control
(Excitation : 405nm, Emission : 460 – 560nm)
The intensity for the fluorescence is further enhanced with respect to 1st and 2nd Trial. The signal peaks at 473nm. A lot of drugs were entering the cell. It may due to the enhancement done to the reaction condition of drugs.
Fig.11(A-C)HK-1 Treatment ; (D-F)HK-1 Control (Excitation : 405nm, Emission : 460 – 560nm)

Fig.11(A-C)HK-1 Treatment ; (D-F)HK-1 Control
(Excitation : 405nm, Emission : 460 – 560nm)


6. MTT assay


Fig.12: 16 samples were used for the MTT Assay and HK-1 was the target cancer cell.
It is found that 12.34% of cells is killed after treatment.
The standard deviation for the result is 0.01259.


7. Emission Spectrum

i. Hep G2:


ii. HK-1:


8. AFM photo

Discussion

1. MAILDI-TOF mass spectrometry
MALDI-TOF mass spectrometry was used to measure the reaction yield of the conjugation between single strand DNA and chlorambucil,
FOLIC ACID or graphite quantum dots. A relatively low yield was found according to the mass spectrum.

In order to obtain a higher product yield, we raised the reaction temperature little by little and attempted 0oC, 25oC, 37oC and 50oC. It is obvious that with the increase of reaction temperature, the product yield also enhances, which can be seen from the mass spectrums. On the other hand, we still raised the reactant ratio between the single strand DNA and molecules from 1:1 to 1:10. However, very little improvement was observed in the yield. That may be because both chlorambucil and FOLIC ACID have a very low solubility in water. So an increase in reactant ratio has less influence on the product yield than in reaction temperature.Here, we present the data under two kinds of reaction condition.

a.The single strand DNA was conjugated with chlorambucil, FOLIC ACID or GQD with 1:1 ratio. And the reaction was undergone at the room temperature about 25 oC.
b.The single strand DNA was conjugated with chlorambucil, folic acid or GQD with 1:10 ratio. And the reaction was undergone at 37 oC.

2. Atomic force spectrometry
The Atomic force microscopy image has a low resolution and the structure of the functionalized DNA origami can hardly be identified which may due to the following reasons:

a.The DNA origami samples is not dried up completely which increase the ADHESIVE force of the AFM instrument
b.The DNA origami samples was clustered on the mica platform
c.The scan size of AFM is restricted from 1 to 5 nm so many area attached with the DNA was not examined properly.

Future Development

1. Modify the DNA origami design with more surface area to accommodate more folic acid, chlorambucil and graphene quantum dot
2. Increase the drug load to enhance the cytotoxicity of the functionalized DNA origami complex

3. Adapt other specific biomarker from cancer cell to increase the targeting efficiency
Personal tools